NetNotes
aſt er a liſt out is completed I will plasma clean for 2-5 minutes prior to insertion; however, someone recently suggested baking our samples for a few hours at 50-100ºC to further reduce contamination. It was also suggested that we place our heater in a bell jar-like setup so that we can pump on the sample during the annealing. I am considering building such a setup, but I would like to see if anyone has constructed something like this before. I would very much appreciate any design suggestions or other tips to help us clean up our samples. Steven R. Spurgeon steven.
spurgeon@pnnl.gov Tue Sep 2 It defi nitely helps to bake—and not be shy about it. 160ºC for 8-12 hours (i.e. overnight) is good. I fi nd that if that doesn’t do it, it won’t come clean. Our setup is not exactly home-made, but it consists of a Pfeiff er HiCube dry pumping station and an MTI quartz tube furnace. T at way it is possible to do vacuum or air or to introduce other gas environments. And the temperature vs. time profi le is programmable. I can send you a picture if you’re interested. May be overkill for you. Larry Scipioni
les@zsgenetics.com Tue Sep 22
Have done this using a turbo vacuum system we used for pumping holders, with a tube connected, covered by a vacuum fl ange with an electrical feedthrough (used parts of an old Penning gauge for that). Inside I clamped a 12 volt DC halogen lamp to produce heat. Our mechanical workshop machined a small open aluminum grid box which holds 16 grids. T is goes into the tube, pump, ramp up the lamp, wait some hours, vent with clean nitrogen. T e small dry turbo system is going to be main cost. T e other parts can be cheap. You might also consider putting a UV source in. Wim Hagen
wim.hagen@me.com Wed Sep 23 I wanted to build a similar system for drying of NMP from my TEM grids. I just hooked up a Peltier cooler/heater with an old desktop PC power supply and controlled it with a mechanical relay, which in turn was controlled by Arduino Uno. Temperature feedback was provided by a LM-35 sensor, which I got in an Arduino starter kit. Peltier heater and sensor was kept in a vacuum oven which had few holes drilled to make way for wires which were then sealed with epoxy resin. It was the cheapest and quickest way to do it and it can easily maintain a temper- ature of 60ºC ± 2º. For higher temperatures and better control, you might try better heating coils, thermocouples and a proportional-integral- derivative (PID) algorithm. T e rest of setup should remain the same. Amit Gupta
amit.welcomes.u@gmail.com Wed Sep 23
Specimen Preparation: holey carbon support fi lm
Has anybody prepared their own holey carbon fi lm supports? We usually use the fi lm on Cu mesh grids. How is the mesh grid manufac- tured? What if I need it on a polymer grid? How do you control the size
of the holes? What is the range of the holes we can achieve? Zhaoxia Zhou
z.zhou@lboro.ac.uk Wed Sep 23
I have done so, and it is trickier than making fi lms without
holes. T ere are several protocols that work, but it may take some time to develop the necessary skills. I found the trickiest part to be the separation of the holey Formvar from the glass slide. Commonly, the oil from the skin of one’s nose is used to coat the slide before dipping in Formvar; however, in my case this oil did not work—it did work for former without holes. A solution of Apiazon L in petroleum ether did work for holey Formvar. Another diffi culty was that separating the fi lm from the slide did not work when the humidity in the lab was high. Bill Tivol
wtivol@sbcglobal.net Wed Sep 23
Years ago, back when dinosaurs still roamed the earth, I made the
holey Formvar fi lms for our lab. We didn’t use carbon fi lms back then in the biological microscopy lab. I used the method described by M. A. Hayat, Principles and Techniques of Electron Microscopy (Biological Applications (Volume 1) , Van Nostrand Reinhold, 1970, 324–332. T e method for production of Parlodian (nitrocellulose) or Formvar (polyvi- nyl formal) plastic fi lms is straightforward but requires practice to learn the technique. T e method is the same for production of continuous and holey Parlodian and Formvar fi lms except that glycerol is added to the Parlodian or Formvar solution prior to casting the fi lms; the glycerol is insoluble in the solvents used to dissolve the plastic fi lms. T e size of holes in in holey fi lms is proportional to the concentration of glycerol in the mixture: 12% (vol/vol) glycerol gives holes with a maximum diameter of approximately 25 μ m whereas 0.08% glycerol gives a maximum hole size of 4 μ m. Although you can learn the method of holey carbon fi lm prepara- tion, if you don’t do it oſt en, the process becomes very laborious and time- consuming. I suggest you consider purchasing your holey carbon fi lm grids from an electron microscopy supply house. T at’s what I did for the last 25 years. T e Hayat book can be found for $8 - $35 on Amazon. Regarding your question about TEM grid manufacture: metallic TEM grids are electroplated. I don’t know how non-metallic grids such as nylon are prepared. Gary M Brown
microscopy.gmb@
gmail.com Wed Sep 23
Specimen Preparation: Lowicryl K4M or HM20
I need to prepare some Lowicryl. I am leaning towards K4M. I have never done this. Can someone give me a few pointers? T e recipe says a brown glass container. But these are pricey containers to purchase for mixing up resin. I would like to use this in my freeze substitution device on some heart tissue for immunolabeling. If I am using ethanol in my resin mixtures, should I use ethanol in my substitution medium instead of acetone? Chris Brantner
chrisbrantner@email.gwu.edu Wed Sep 30
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